We are using a site-directed mutagenesis approach to define structure/function relationships in an effort to determine parameters affecting polymerization, solubility and stability of Hb S in vitro. We have assembled hemoglobins with Ala, Val, Leu and Trp at the beta6 position employing a bacterial expression system and showed that polymer interactions between the the beta6 position of one tetramer and the U-shaped EF helix region on another are greatly influenced by hydrophobicity and stereospecificity of the amino acid side chain at the beta6 position in deoxyhemoglobin. Strong hydrophobic amino acids, in addition to the shape, size and position of the side chair in hemoglobin lead to enhanced polymerization of tetramers in vitro, and also result in decreased solubility. These results suggest that modified hemoglobins can be engineered with altered biochemical properties and that these can be used as tools for structure/function studies for use in defining parameters affecting polymerization, solubility, oxygen binding and stability of hemoglobin molecules. We have also implemented a recently described expression system in which alpha- and beta-globin chains are coexpressed in yeast (S. cerevisiae) and form soluble tetrameric hemoglobin. The alpha- and beta chains assemble into tetramers by incorporating endogenous heme which results in high level production of soluble tetramers. This system will facilitate production of large amounts of assembled tetramers for a variety of studies proposed in this grant. Specific Aims include the following: (1) We will prepare various Hb mutants at beta22, beta23, beta121, alpha6 and alpha23 to define what role these sites play in facilitating polymerization of Hb S. These studies should help clarify the role that various important tetramer contact sites, other than the beta6 position, play in polymerization, solubility and tetramer stability. Identification of amino acids which accelerate polymerization may also help facilitate production of a transgenic animal model for sickle cell disease. (2) We will study the relationship between oxygen affinity and polymerization by engineering Hb mutants at beta23 and beta121, since some naturally-occurring variants at these positions are known to alter 02 affinity. (3) We will also evaluate the effects of pH on polymerization by preparing Hb substitutions at His beta116 and His beta117 which are involved in a contact site on Hb S polymers. (4) Finally, we will continue studies aimed at understanding the molecular basis for inhibition of Hb S polymerization by Hb F. We will use recombinant Hb F and Hb A modified at the gamma87 and beta87 positions, respectively, to study the inhibitory effects on polymerization of Hb S in vitro.